A Rapid Method for Isolation of Double Stranded RNA

A rapid and simple method for the isolation and purification of dsRNA is presented. The crucial step of this method is the
extraction of proteins and DNA with acid phenol. After the extraction, only RNA is left in the aqueous phase. ssRNA
contamination of the RNA preparation can be greatly reduced when ammonium sulfate is present during the extraction.

INTRODUCTION

Double-stranded RNA can be isolated from cells and tissues by several different methods. Most often, total nucleic acid
extracts from cells or from dsRNA-enriched fractions of cell homogenates are fractionated either by chromatography [1] or
by selective precipitation with 4 M LiCl [2]. Both methods are time-consuming and often result in large losses of material.

We developed a one-step method to isolate and purify dsRNA from the protozoon Trichomonas vaginalis. DNA and
proteins are extracted from the cells with phenol pH 4.0, which is followed by precipitation of dsRNA from the aquaeous
phase with isopropanol. The method is rapid, inexpensive and gives very good yields of dsRNA. We showed that this
approach was also suitable for the isolation of dsRNA from nonprotozoal material.

All steps of isolation were performed at room temperature. Trichomonads in growth medium ta tryptose, yeast extract,
maltose medium (TYM) [4] with or without agarose) or in 0.8' NaCl (5-400 million cells/ml) were transferred to a plastic
centrifuge tube, i volume of phenol pH 4.0 water-saturated phenol equilibrated with 50 mM acetate buffer pH 4.C) was
added, and the tube was ' tightly capped and vigorously;. shaken for 3 minutes. The phenol and aqueous phases were
separated by centrifugation and the agueous phase was reextracted with 8 volumes of chloroform-isoamyl alcohol t4:11.
After centrifugation the upper phase was transferred to a new tube, a ).75 volume of isopropanol was added, and nucleic
acids were precipitated from the mixture by three cycles of freeing (in dry ice or liquid nitrogen) and thawing,. The
precipitated RNA was collected by centrifugation, the pellet was washed with 60% ethanol, dried and dissolved in TE
buffer 10 mM Tris, 1 mM EDTA, pH 8.0).

Isolation of DNA and RNA with phenol pH 8.0,

The same method as described above for dsRNA was employed, but phenol pH 8.0 (water-saturated phenol equilibrated
with at first 1 M and then 0.05 M Tris-HCl pH 8.0)) instead of phenol pH 4.0 ) was used for extraction.

Isolation of DNA and RNA with chloroform.

Trichomonads in 0.8% NaCl (5-100 million cells/ml ) were lysed with guanidine hydrochloride or guanidine
iscthiocyanate (final concentration 4 M, stock solutions 8 M and 6 M, respectively). The extraction was performed with
two:.volumes of chloroform-isoamyl alcohol (24:lI 5 times and nucleic aciids were precipitated as described above.

RESULTS AND DISCUSSION

As revealed by electrophoretic patterns of nucleic acids extracted from equal numbers of T. vaginalis cells by three
different methods (Fig. 1), extraction with acid phenol gave the highest yields of dsRNA without any apparent DNA
contamination.

Solubility of the nucleic acid pellet was also studied. TE buffer was added to the pellets and samples were taken
sequentially in the course of i20 minutes as specified in the legend to Fig. 2. At the end of the experiment the samples were
electrophoresed (Fig. 2). The dsRNA extracted by acid phenol dissolved completely in 30-60 minutes; dsRNA extracted
with guanidine hydrochloride needed 120 minutes to dissolve. The guanidine isothiocyanate method gave a very poor yield
of dsRNA.

Comparison of the electrophoretic patterns of dsRNA extracted from T. vaginalis with phenol (pH 4.4 as well as pH 8.0)

and with guanidine hydrochloride tor guanidine isothiocyanate) showed that one dsRNA zone was absent in the f ormer
(Fig. 1). A similar effect was observed when

Dissolution of nucleic acids pellets. Pellets of nucleic acids isolated from 0 million trichomonads with acid phenol Ia),
guanidine hydrochloride (b, or guanidine isothiocyanate tc were dissolved in TE buffer for 15, 30, 60, or 120 minutes. The
arrows point to the ones of DNA (near the start of electrophoresis) and RNA.

dsRNA had been extracted from the yeast Saccharomyces cerevisiae. In this case the extraction was started from
SDS-lysed protoplasts (prepared according to Eddy and Williamson [6]). Figure 3 shows that the L species of dsRNA
(molecular mass 3.2 megadaltons [7] was extracted with phenol far more effectively and the M species (molecular mass
1.0-1.3 megadaltons [8]) far less effectively than by the guanidine hydrochloride method. This phenomenon could have
been due to the presence of proteins covalently bound to these dsRNAs, which had made them enter the phenolic phase or
the protein interphase [9].

The acid phenol method of isolation of dsRNA resembled a hot phenol method for the isolation of ssRNA C147; both
extraction procedures eliminate the DNA into the phenol phase, leaving the RNA in the aqueous phase. Our results showed
that the acid phenol method could be used for isolation of ssDNA and appeared to be even more effective than the hot
phenol method. which must be repeated several times to achieve 95% elimination of DNA [10,11]. The quality of the
ssRNA obtained is under investigation now, because of the reported depurination of nucleic acids at low pH [12] and
elimination and partial degradation of poly(A)RNA with phenol [11, 13]. We observed that when a certain amount of
ammonium sulfate (not NaCl or KCl) was added to the extraction mixture (Fig. 4), most of the ssRNA could enter the
phenol phase. (For details see the legend to the figure). When high concentrations of ammonium sulfate were used, the
water phase turned denser than the phenol phase and accumulated at the bottom of the centrifuge tube. Moreover, during
isopropanol precipitation of dsRNA two distinct phases. an isopropanol and an ammonium sulfate-saturated water phase,
appeared. In that case ammonium sulfate had to be extracted from the lower aquaeous phase with 60% ethanol before a
pellet of RNA could be obtained by centrifugation.

Phenol extraction usually removes proteins, leaving DNA and RNA in the aqueous phase. It has been demonstrated,
however, that under conditions of a particular pH, ionic strength or

FIGURE 4

Elimination of ssRNA contaminant by ammonium sulfate. Before the extraction the samples (aliquots of trichomonads in
0.8% NaCl) were saturated to 20, 30, 40, 50 and 60% with ammonium sulfate (solid) (lanes 1-5, respectively).

temperature, it removes DNA and some classes of RNA with the proteins. Several methods of isolation of total ribonucleic
acids [14], mRNA [11, 15, 16], rRNA [10] or covalently closed circular DNA (cccDNA) [17] are based on this
phenomenon, the physical basis of which is not fully understood. In many systems a coprecipitation of DNA or
poly(A)RNA with denatured proteins or with crystals of potasium dodecylsulphate is considered to be the most probable
explanation [10, 11]. In the case of our method, as well as that of Zasloff et al. [17] (isolation of covalently closed circular
DNA), however, a different mechanism must be operating, because DNA can be selectively extracted into the phenol
phase from protein-free solutions. Both methods share the acid phenol extraction step. Zasloff et al [17] demonstrated that
when phenol pH 4.5 instead of pH 4.0 was used, the DNA stayed in the aqueous phase. The methods differ, however, in
their sensitivity to variation of ionic strength. When Zasloff et al extracted NA from buffers containing more than 200 mM
NaCl, all nucleic acids entered the phenol phase (their material did not contain dsRNA). Our results showed that isolation
of dsRNA is insensitive to an extensive variation of the concentration of ammonium sulfate (Fig. 4), NaCl or KCl.

From a practical point of view, the most important difference between our and Zasloff's method is that dsRNA can be
isolated directly from cell suspensions while the isolation of covalently closed circular DNA requires previous elimination
of proteins by extraction with phenol pH 8. It follows that using Zasloff's method, dsRNA, if present, could contaminate
cccDNA extracts, while when isolating dsRNA by our method, no contamination of dsRNA extracts by cccDNA can be
expected.

ACKNOWLEDGMENT

I thank Dr. J.Čerkasov (Charles University, Prague) for his advice and aid and Drs. J.Kulda (Charles Univereity) and
M.Muller (Rockefeller University, New York) for reading and commenting on the manuscript.